【作者】 吴彦瑞；

【导师】 范明； 范文红；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2010， 博士

【摘要】 Copines蛋白家族是近期发现的一类进化保守的Ca2+依赖性磷脂结合蛋白,该家族蛋白有两个保守结构域:N端两个C2 domain,是结合钙和磷脂的部位,已知蛋白激酶C、磷脂酶C和突触结合蛋白等也有该结构域;C端的A domain,类似于整合素(integrin)中的A domain,具有蛋白质相互作用位点,是结合靶蛋白的结构域。CPNE5定位于第6号染色体的6p21.1基因座,含一个1782bp的开放阅读框,编码593个氨基酸的蛋白。本研究主要围绕CPNE5对TNF-a诱导的NF-kB转录激活活性的影响及其作用机制,以及CPNE5在细胞凋亡方面的作用进行了研究。在HEK293细胞中,TNF-a诱导CPNE5 mRNA表达显著上调。含有NF-κB调控靶序列的双荧光素酶报告基因实验结果表明,外源表达CPNE5能够抑制TNF-a诱导的NF-κB的转录激活活性。RT-PCR实验显示,过表达CPNE5抑制了NF-κB下游基因Bcl-2等在核酸水平的表达。为研究CPNE5保守区域A domain在CPNE5抑制NF-κB活性中的作用,我们构建了C2结构域缺失的AD(A domain)的GFP表达载体,发现AD-GFP融合蛋白在细胞内呈弥散表达,不同于CPNE5的定位。我们发现AD的高表达,可以显著地抑制TNF-a所诱导的NF-κB转录激活活性,且这种抑制作用与AD转染量具有剂量依赖性。这说明CPNE5的AD结构域在其抑制NF-κB转录激活中起到重要作用。在发现CPNE5可以显著抑制TNF-a对NF-κB的转录激活的基础上,我们进一步对抑制机制进行了研究。首先我们在HEK293细胞中建立了CPNE5的四环素诱导表达系统(Tet-on)。经过Zeocin筛选,我们挑取了7个pcDNATM 4/TO-CPNE5 T-Rex-293细胞克隆株。这些细胞株经四环素诱导12h~24h后,目的蛋白CPNE5有不同程度的表达。我们选取诱导表达量较高的细胞株,研究高表达CPNE5抑制NF-κB转录激活的机制。Western-blot结果显示,CPNE5高表达并不影响NF-κB亚单位p65的入核。细胞免疫荧光实验结果也验证了这一结果,正常HEK293细胞中,TNF-a刺激能诱导NF-κB的快速入核。Western blot实验和细胞免疫荧光实验结果均表明,外源高表达CPNE5没有阻止TNF-a诱导的NF-κB入核。这些结果表明,CPNE5不通过NF-κB出入核调节机制来抑制NF-κB的活性。凝胶阻滞实验结果表明,CPEN5过表达抑制了NF-κB的DNA结合活性。免疫共沉淀(co-IP)实验结果表明,外源表达的CPNE5和NF-κB(P65)有相互作用。为研究CPNE5是否如CPNE1一样可以剪切P65或P50,我们过表达CPNE5后再进行TNF-a处理,Westernblot检测未发现P65/P50有剪切体出现,这表明CPNE5没有如同家族的CPNE1一样诱导P65或P50的剪切。为研究外源表达CPNE5是否因为抑制了NF-κB的活性,从而抑制了TNF-a诱导的与细胞生存相关的信号通路,进而促进了TNF-a诱导的细胞凋亡,我们研究了外源表达CPNE5对TNF-a诱导的细胞凋亡的影响。与空载体转染组相比,外源表达CPNE5的HEK293细胞,在TNF-a作用后,细胞形态学上出现特征性的凋亡改变,如细胞外形模糊、细胞皱缩、细胞体积缩小等。流式细胞术检测结果显示,TNF-a处理12h或24h时,实验组细胞凋亡率高于对照组。分析介导凋亡的信号分子发现,凋亡核心成员半胱氨酸-天冬氨酸蛋白酶(caspase)的切割底物PARP参与了此过程。Western Blot结果表明,外源表达CPNE5促进了PARP的剪切。以上结果表明,CPNE5可以在一定程度上促进TNF-a诱导的细胞凋亡。而外源表达CPNE5对CHX和TNF-a(-/+)诱导的细胞凋亡无显著的影响。CHX是一种真核细胞蛋白翻译的常见抑制剂,它可以通过抑制核糖体亚基肽转移酶活性来阻止蛋白质的合成。以上实验结果表明CPNE5和CHX促进TNF-a诱导细胞凋亡的效应有重叠,这提示二者促凋亡机制可能有类似之处,即通过抑制抗凋亡蛋白的合成促进凋亡。我们在检测NF-κB下游基因时,发现一个有趣的现象:无TNF-a诱导时,过表达CPNE5能够显著增强NF-κB下游基因Bcl-xL的蛋白表达水平。此时,NF-κB的转录调控活性并没有被激活。Bcl-xL属于Bc1-2家族I类成员,具有抑制细胞凋亡的作用,已有报道表明Bcl-xL也参与了TNF-a对NF-κB活性的调控。进一步调研文献发现,Bcl-xL的表达除了受NF-κB调控外,还有很多因子可以调控它的表达,其中重要的如STATs和Ets等。由此,我们推测过表达CPNE5可能通过一条不依赖NF-κB活性的信号通路促Bcl-xL表达,而正由于Bcl-xL这种表达水平的升高,导致了尽管CPNE5有效地抑制了NF-κB的转录激活活性,却依然没有显著促进NF-κB诱导的细胞凋亡。为验证这一设想,我们钓取了人Bcl-xL基因并将其构建到真核表达载体。双荧光素酶报告基因实验结果显示,过表达Bcl-xL能够抑制NF-κB转录激活活性;转染Bcl-xL的RNA干涉片段SiRNA-BclxL,降低了Bcl-xL的表达同时弱化CPNE5对NF-κB活性的抑制,表明Bcl-xL可能在CPNE5抑制NF-κB转录激活活性过程中发挥重要的作用。本研究内容概括为:1.外源表达CPNE5抑制了TNF-a诱导了NF-κB的转录激活活性;CPNE5的A结构域也有这种抑制作用,并且呈现剂量依赖性;2. CPNE5不影响p65的出入核,表明CPNE5不通过NF-κB出入核调节机制来抑制NF-κB的活性,但CPEN5抑制了NF-κB与DNA结合的活性,且可以和P65相互作用;3. CPNE5促进了TNF-a诱导的凋亡率增加;但对CHX或TNF-a/CHX诱导的细胞凋亡无影响;4.成功钓取Bcl-xL基因;发现CPNE5表达诱导Bcl-xL表达水平上调;过表达Bcl-xL抑制NF-κB活性,SiRNA-BclxL弱化CPNE5对NF-κB活性的抑制,表明Bcl-xL可能参与CPNE5抑制NF-κB活性的过程。我们的研究首次证明了CPNE5及其保守结构域AD对NF-κB的转录激活具有抑制作用,其机制为CPNE5抑制NF-κB DNA结合活性,且可能和CPNE5与P65的结合有关。我们也研究了CPNE5对细胞凋亡的影响。过表达CPNE5抑制NF-κB的活性且促进凋亡。尽管过表达CPNE5也会上调Bcl-xL的表达,但在整个TNF-a诱导的细胞凋亡过程中,过表达CPNE5总体上呈现促凋亡的效应。更多还原

【Abstract】 The copines are a novel family of ubiquitous Ca2+-dependent, phospholipid-binding proteins. Copines are comprised of two N-terminal C2 domains and a C-terminal A domain (AD). The C2 domains of copines are responsible for calcium and phospholipid-binding. C2 domains also present in proteins such as protein kinase C, phospholipase C and synaptotagmin. Because of A domain, copines may also be involved in protein-protein interactions since they contain a domain similar to the protein-binding‘A domain’of integrins. CPNE5 is mapped on chromosome 6p21.1. CPNE5 contains a complete open reading frame of 1782bp which encoding a 593 amino acid protein. Here, we show the studies on CPNE5 gene function, including its regulation in TNF-a induced NF-kB transcriptional activation and the possible mechanisms, and its role in apoptosis.In HEK293 cells, TNF-a treatment significantly increased CPNE5 mRNA expression. Dual-Luciferase Reporter Assay showed that exogenous expressed CPNE5 inhibited the transcriptional activity of NF-κB induced by TNF-a. RT-PCR results showed that overexpression of CPNE5 inhibited mRNA expression of NF-κB downstream genes such as Bcl-2. To investigate the role of C2 domains in the inhibition of NF-κB transcriptional activity, we constructed an AD-GFP expression vector with two C2 domain deletion. We found high expression of AD can significantly inhibit the transcriptional activity of NF-κB induced by TNF-a. The inhibition is dose-dependent with the amount of AD transfection. This indicates that AD domain plays an important role during the inhibition of NF-κB transcriptional activity.To further investigated the inhibitory mechanism, we established the tetracycline induced expression system (Tet-on) of CPNE5 in HEK293 cells. After Zeocin selection, we picked seven pcDNATM 4/TO-CPNE5 T-Rex-293 cell clone. CPNE5 was differentially expressed after 12h induction by tetracycline in these cells. We used the cell clones with the highest CPNE5 expression level to investigate the mechanism of CPNE5 inhibiting NF-κB activity. The translocation of NF-κB subunit P65 from cytoplasm to nucleus is the typical activation of NF-κB. We first detected whether CPNE5 overexpression inhibit the translocation of P65. Both western blot and imunofluorescence results showed that CPNE5 overexpression had no effect on translocation of P65 induced by TNF-a. These results indicate that CPNE5 did not suppress NF-κB transcriptional activity through the classical NF-κB regulatory pathway. However, electrophoretic mobility shift assay (EMSA) result showed that CPEN5 overexpression repressed NF-κB DNA-binding activity. Immunoprecipitation experiments showed that exogenous expressed CPNE5 interact with NF-κB (P65) directly.TNF-a induced activation of NF-κB plays important role in cell survival. To study whether exogenous expression of CPNE5 induces apoptosis through inhibiting the transcriptional activity of NF-κB, we explored the effect of exogenous CPNE5 on apoptosis induced by TNF-a. HEK293 cells were transfected with pcDNA3.1 and pcDNA3.1-CPNE5 respectively and treated with TNF-a, the apoptosis rate were detected by FACS analysis. Compared to the cells transfected with pcDNA3.1, cells transfected with pcDNA3.1-CPNE5 showed higher apoptsis rate. The cells expressing exogenous CPNE5 showed characteristic morphological changes of apoptosis, such as cell shape fuzzy, cell shrinkage, reduced cell volume and so on. Further investigation into the apoptosis signaling showed that PARP, a cutting substrate of caspase, is involved in the above process. Western blot result showed that exogenous expression of CPNE5 promoted PARP cleavage. These results showed that CPNE5 could promote TNF-a induced apoptosis to some degree. Exogenous expression of CPNE5 had no significant effect on CHX (TNF-a–/+) induced apoptosis. CHX is a most common inhibitor of eukaryotic protein translation, which can inhibit the 60S ribosomal subunit peptide transferase activity to prevent protein synthesis. These results showed that exogenous expression of CPNE5 regulated TNF-a induced apoptosis process involved with synthesis of protective protein.Further investigation of the expression of NF-κB downstream genes, we found an interesting result. Without TNF-a induced activation of NF-κB, overexpressed CPNE5 upregulate Bcl-xL expression significantly. Bcl-xL, a member of the Bcl-2 family, is also known to inhibit TNF-a-induced apoptosis. We cloned the cDNA and constructed the eukaryotic expression vector of Bcl-xL. Overexpression of Bcl-xL inhibited the transcriptional activity of NF-κB. Downregulate the expression of Bcl-xL with SiRNA-Bcl-xL weaken the inhibition of NF-κB activity by CPNE5. These results indicate that Bcl-xL may play an important role in inhibition of NF-κB transcriptional activation by CPNE5. This research summarized as: 1. exogenous expression of CPNE5 inhibited the TNF-a induced NF-κB transcriptional activity; A domain of CPNE5 also had this inhibitory effect in dose-dependent manner; 2. CPNE5 had no effect on translocation of P65; CPNE5 did not suppress NF-κB transcriptional activity through the classical NF-κB regulatory pathway, but CPEN5 inhibited DNA binding activity of NF-κB and interacted with P65; 3. CPNE5 promoted TNF-a induced apoptosis but had no effect on apoptosis induced byCHX or TNF-a/CHX; 4. overexpressed CPNE5 upregulate Bcl-xL expression; overexpression of Bcl-xL inhibited NF-κB activity; SiRNA-BclxL weaken the inhibition of NF-κB activity by CPNE5, these indicated that Bcl-xL may be involved in inhibition of NF-κB activity by CPNE5.Our results for the first time revealed that CPNE5 inhibit NF-κB transcriptional activity. CPNE5 might block the DNA binding activity of NF-κB through interacting with p65. In addition, we also found that overexpression of CPNE5 enhance cell apoptosis induced by TNF-a. The upregulation of Bcl-xL expression by CPNE5 might play a certain role in this process.更多还原